Oxytocin increases invasive properties of endometrial cancer cells through phosphatidylinositol 3-kinase/AKT-dependent up-regulation of cyclooxygenase-1, -2, and X-linked inhibitor of apoptosis protein.

Traditionally, oxytocin (OT) is well known to play a crucial role in the regulation of cyclic changes in the uterus, implantation of the embryo, and parturition. Recently, an additional role for OT has been identified in several types of cancer cells in which OT acts as a growth regulator. In endometrial cancer cells, OT is known to efficiently inhibit cellular proliferation. In the present study, we show that OT increases invasiveness of human endometrial carcinoma (HEC) cells, which are otherwise resistant to the growth-inhibiting effects of OT. Using pharmacological inhibitors, invasion assay, RNA interference, and immunofluorescence, we found that OT enhances the invasive properties of HEC cells through up-regulation of X-linked inhibitor of apoptosis protein (XIAP), matrix-metalloproteinase 2 (MMP2), and matrix-metalloproteinase 14 (MMP14). In addition, we show that OT-mediated invasion is both cyclooxygenase 1 (PTGS1) and cyclooxygenase-2 (PTGS2) dependent via the phosphatidylinositol 3-kinase/AKT (PIK3/AKT) pathway. PTGS2 knockdown by shRNA resulted in XIAP down-regulation. We also show that OT receptor is overexpressed in grade I to III endometrial cancer. Taken together, our results describe for the first time a novel role for OT in endometrial cancer cell invasion.

Characterization of EN-1078D, a poorly differentiated human endometrial carcinoma cell line: a novel tool to study endometrial invasion in vitro.

BACKGROUND: To date, tools to study metastasis in endometrial cancers are insufficiently developed. The aim of this study was to characterize the cell line EN-1078D, a new endometrial carcinoma cell line derived from a metastasis to the ovary. METHODS AND RESULTS: Cells were characterized using cytology, transmission electron microscopy, karyotyping and morphological appearance in culture. Molecular features were determined by RT-PCR, Western Blot, FISH and sequencing. MTT proliferation assays were performed to investigate the sensitivity of EN-1078D to anticancer agents such as cisplatin and doxorubicin. Also, subcutaneous and intravenous injections in nude mice were done to test the tumorigenic and metastatic properties of EN-1078D cells. Our results indicate that EN-1078D cells express both oestrogen receptors isoforms (ER alpha and ER beta) and also low levels of progesterone receptor B (PR-B). In addition, this cell line expresses high levels of MMP-2 and MMP-14 mRNA, low levels of TIMP-1 and TIMP-2 transcripts and no detectable levels of MMP-9 mRNA. Moreover, all nude mice developed tumors by subcutaneous injections and cell invasion was observed in vitro in response to TGF-beta 3. Her-2/neu was not overamplified but mutations in the C-2 domain of PTEN gene as well as codon 12 of the K-Ras gene were found. Finally, EN-1078D shows sensitivity to drugs commonly used in chemotherapy such as cisplatin and doxorubicin: IC50 of 2.8 microM of cisplatin after 72 hours of exposure and 0.54 microM of doxorubicin after 48 hours. CONCLUSION: Taken together, these results suggest that EN-1078D will be an excellent tool to study the properties of metastatic endometrial cancer cells in vitro and their regulation by sex steroids.

Effects of 4-hydroxytamoxifen, raloxifene and ICI 182 780 on survival of uterine cancer cell lines in the presence and absence of exogenous estrogens.

The impact of 17beta-estradiol and antiestrogens on uterine cancer cells is poorly understood. The aim of this study was to determine the impact of 17beta-estradiol, 4-hydroxytamoxifen, raloxifene and ICI 182 780 on the cell proliferation of six uterine cancer cell lines: HeLa, HEC-1-A, KLE, RL-95-2, Ishikawa and EN-1078D. The effects of these compounds on the cell proliferation of the six uterine cancer cell lines were studied in the presence and absence of estrogens (phenol red and serum deprivation of sex steroids). In a general manner, 17beta-estradiol and 4-hydroxytamoxifen showed similarities in their effects whereas raloxifene showed a different pattern of cell proliferation (agonistic and antagonistic) and ICI 182 780 had antagonistic activity. In the presence and absence of estrogens, we observed that each cell line had diverse expression of ERalpha, ERbeta, GPR30 and REA. GPR30 mRNA expression was significantly reduced in a serum/phenol-free medium. REA mRNA expression was not influenced by the media. Results demonstrated the importance of removing phenol red and the use of deprived serum when studying uterine cancer cells in relationship with 17beta-estradiol and antiestrogens. The affinity of each compound to the binding of ERalpha and ERbeta was very similar with the exception of raloxifene that had a preference for ERalpha binding. Akt phosphorylation/activity was reduced in cells cultured in a phenol red- and steroid-free culture medium indicating that the presence of steroids in the culture media can influence the activity of this survival pathway. Our results suggest that the expression of ERalpha, ERbeta and GPR30 are influenced by sex steroids and might play a role in the response of cells to 17beta-estradiol and antiestrogens but are not the only factors involved in this process.

TGF-beta expression during rat pregnancy and activity on decidual cell survival.

BACKGROUND: During early rat pregnancy, trophoblast of the tiny embryo joins with the endometrium and epithelial cells undergo apoptosis. Near the end of pregnancy, regression of the decidua basalis (DB) is also observed (from day 14 to 20). However, little is known about the intra-cellular and molecular mechanisms involved in apoptosis regulation in the uterus during pregnancy. The objective of the present study was to investigate the presence and the developmental expression of transforming growth factor-beta isoforms (TGF-beta well known differentiation factor) in the rat endometrium throughout pregnancy and its action in vitro using cultured endometrial stromal cells. METHODS: In vivo: Rats were killed at different days of pregnancy (days 2-20) and uteri removed to collect endometrial protein extracts or the uteri were fixed, embedded and sectioned for immunohistochemistry (IHC) and in situ cell death analyses using TdT-mediated dUTP nick end labeling (TUNEL). In vitro: Rats were ovariectomized and decidualization was induced using sex steroids. Endometrial stromal decidual cells were then collected and cultured. RESULTS: An increase of apoptosis in the DB on days 14, 16 and 18 was observed. Cleaved caspase-3 was clearly detected during regression of the DB by Western analysis and immunofluorescence. Western analyses using endometrial protein extracts demonstrated that TGF-beta1, TGF-beta2 and TGF-beta3 were highly expressed at the time of DB regression (day 14). During early pregnancy, TGF-beta1 and -beta2 expressions raised at days 5.5 to 6.5. TGF-beta3 protein was not detected during early pregnancy. IHC analyses revealed that TGF-beta1 and -2 were found surrounding both epithelium (luminal and glandular) in the stroma compartment at the implantation site, and TGF-beta3 was mainly located surrounding endometrial epithelium in the stroma compartment. Smad2 phosphorylation was increased at the time of DB regression. In vitro studies using decidual endometrial stromal cells revealed that TGF-beta1 induced apoptosis and Smad2 phosphorylation. Moreover, TGF-beta1 reduced both Akt (a well known survival factor) phosphorylation and XIAP (X-linked inhibitor of apoptosis protein) expression in decidual endometrial stromal cells in vitro. CONCLUSION: Taken together, these results suggest that TGF-beta isoforms are regulated differently during pregnancy and may have an important role in the control of apoptosis and cell survival at specific stages during pregnancy.

Opposite regulation of XIAP and Smac/DIABLO in the rat endometrium in response to 17beta-estradiol at estrus.

During rat estrous cycle, the endometrium proliferates in response to sex steroids and specific endometrial epithelial cells undergo apoptosis in absence of embryonic factors. The central executioner of apoptosis is a family of aspartic acid-specific cysteine proteases known as caspases. Smac/DIABLO is released from the mitochondria during apoptosis and its stimulation promotes caspases activation by neutralizing members of the inhibitor of apoptosis proteins (IAPs) family, such as X-linked inhibitor of apoptosis protein (XIAP). The aim of this study was to investigate the involvement of Smac/DIABLO and XIAP in the control of caspases activation in endometrium of cycling rats. Polyoestrus female rats were sacrificed at each stage of estrous cycle (diestrus, proestrus, estrus, and metestrus). Endometrial protein extracts were collected to perform Western Blot analysis. Alternatively, uterine horns were sectioned for immunohistochemistry (IHC). We and others showed previously the presence of apoptosis at estrus in rat uterine epithelium. In the present study, cleaved caspase-3, -6, and -7 fragments were detected at estrus. IHC confirmed that caspase-3 was present only in luminal and glandular epithelium at estrus. XIAP was highly expressed at estrus in both epithelial and stromal cells. In contrast, expression of Smac/DIABLO was elevated at diestrus, proestrus and metestrus but was minimal at estrus. Treatment of ovariectomized rats with 17beta-estradiol induced XIAP expression and inhibited Smac/DIABLO protein expression in the endometrium. Cleaved caspase-3, -6, and -7 fragments increased in endometrial protein extracts following 17beta-estradiol treatment. Expression of NF-kappaB and IkappaB proteins, and IkappaB phosphorylation status were detected in the endometrium but were not influenced by the estrous cycle. These findings suggest that Smac/DIABLO and XIAP are regulated differently and may play important roles in the regulation of endometrial cell fate. Moreover, this study confirms a key role for executioner caspases in the control of apoptotic processes at estrus in the rat uterus.

Regulation of Akt expression and phosphorylation by 17beta-estradiol in the rat uterus during estrous cycle.

Molecular and intra-cellular mechanisms involved in the regulation of apoptosis processes in endometrial cells are poorly understood and documented. We have investigated the possibility that Akt survival pathway might be involved in the regulation of apoptosis in the uterus during the estrous cycle. Rats with regular estrous cycle (4 days) were killed at different days of estrous cycle (diestrus, proestrus, estrus and metestrus). Uteri were collected and fixed for immunohistochemical staining (IHC) and apoptotic cell death detection by [TdT]-mediated deoxyuridinetriphosphate nick end-labelling (TUNEL) or endometrial protein extracts collected for Western analysis. TUNEL analysis revealed that apoptosis was mainly found at estrus compared to other day of estrous cycle. TUNEL positive cells were apparent in luminal epithelial cells only. No apoptotic cells were observed at proestrus. In contrast, proliferation was maximal at proestrus as confirmed with the expression of CDC47/MCM7 (a cell proliferation marker). Intact form of caspase-3 was maximal at proestrus and was reduced only at estrus. Likewise, presence of a specific cleaved caspase-3 fragment was observed only at estrus and IHC revealed that cleaved caspase-3 signal was found in luminal epithelial cells. PTEN protein, a phosphatase involved in the regulation of Akt phosphorylation, was present at all days of estrous cycle and showed no significant regulation in relation to cycle. Expression of phospho-Akt (the activated form of Akt) was present at metestrus, diestrus, and proestrus but decreased significantly at estrus. Akt protein expression was maximal at estrus. IHC revealed that Akt expression was high in both stromal and epithelial cells at estrus. Further studies using ovariectomized rats demonstrated that 17beta-estradiol increased endometrial cell proliferation which was accompanied by an increase of both Akt expression and phosphorylation. These results suggest that increased Akt expression and activity in response to estradiol may be an important mechanism to protect endometrial cells from apoptotic triggering and to induce endometrial cell proliferation, whereas inhibition of Akt activity leads to caspase-3 activation and apoptosis in endometrial cells.